+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-40668 | |||||||||
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Title | Class4 80S with only 60S | |||||||||
Map data | Consensus map | |||||||||
Sample |
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Keywords | Stalled polysomes / puromycin / neuronal RNA granule / Cryo-EM / ribosome. / RIBOSOME | |||||||||
Biological species | Rattus (rat) | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 3.1 Å | |||||||||
Authors | Ortega J | |||||||||
Funding support | Canada, 1 items
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Citation | Journal: Proc Natl Acad Sci U S A / Year: 2024 Title: Puromycin reveals a distinct conformation of neuronal ribosomes. Authors: Mina N Anadolu / Jingyu Sun / Jewel T-Y Li / Tyson E Graber / Joaquin Ortega / Wayne S Sossin / Abstract: Puromycin is covalently added to the nascent chain of proteins by the peptidyl transferase activity of the ribosome and the dissociation of the puromycylated peptide typically follows this event. It ...Puromycin is covalently added to the nascent chain of proteins by the peptidyl transferase activity of the ribosome and the dissociation of the puromycylated peptide typically follows this event. It was postulated that blocking the translocation of the ribosome with emetine could retain the puromycylated peptide on the ribosome, but evidence against this has recently been published [Hobson , , e60048 (2020); and Enam , , e60303 (2020)]. In neurons, puromycylated nascent chains remain in the ribosome even in the absence of emetine, yet direct evidence for this has been lacking. Using biochemistry and cryoelectron microscopy, we show that the puromycylated peptides remain in the ribosome exit channel in the large subunit in a subset of neuronal ribosomes stalled in the hybrid state. These results validate previous experiments to localize stalled polysomes in neurons and provide insight into how neuronal ribosomes are stalled. Moreover, in these hybrid-state neuronal ribosomes, anisomycin, which usually blocks puromycylation, competes poorly with puromycin in the puromycylation reaction, allowing a simple assay to determine the proportion of nascent chains that are stalled in this state. In early hippocampal neuronal cultures, over 50% of all nascent peptides are found in these stalled polysomes. These results provide insights into the stalling mechanisms of neuronal ribosomes and suggest that puromycylated peptides can be used to reveal subcellular sites of hybrid-state stalled ribosomes in neurons. | |||||||||
History |
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-Structure visualization
Supplemental images |
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-Downloads & links
-EMDB archive
Map data | emd_40668.map.gz | 188.3 MB | EMDB map data format | |
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Header (meta data) | emd-40668-v30.xml emd-40668.xml | 12.3 KB 12.3 KB | Display Display | EMDB header |
FSC (resolution estimation) | emd_40668_fsc.xml | 14.1 KB | Display | FSC data file |
Images | emd_40668.png | 94 KB | ||
Filedesc metadata | emd-40668.cif.gz | 3.6 KB | ||
Others | emd_40668_half_map_1.map.gz emd_40668_half_map_2.map.gz | 190.7 MB 190.7 MB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-40668 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-40668 | HTTPS FTP |
-Validation report
Summary document | emd_40668_validation.pdf.gz | 915.4 KB | Display | EMDB validaton report |
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Full document | emd_40668_full_validation.pdf.gz | 915 KB | Display | |
Data in XML | emd_40668_validation.xml.gz | 21.6 KB | Display | |
Data in CIF | emd_40668_validation.cif.gz | 28.2 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-40668 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-40668 | HTTPS FTP |
-Related structure data
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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-Map
File | Download / File: emd_40668.map.gz / Format: CCP4 / Size: 240.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||
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Annotation | Consensus map | ||||||||||||||||||||
Voxel size | X=Y=Z: 1.09 Å | ||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||
Details | EMDB XML:
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-Supplemental data
-Half map: Consensus map half map 1
File | emd_40668_half_map_1.map | ||||||||||||
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Annotation | Consensus map half map 1 | ||||||||||||
Projections & Slices |
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Density Histograms |
-Half map: Consensus map half map 2.
File | emd_40668_half_map_2.map | ||||||||||||
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Annotation | Consensus map half map 2. | ||||||||||||
Projections & Slices |
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Density Histograms |
-Sample components
-Entire : Class4_80S_with_only_60S
Entire | Name: Class4_80S_with_only_60S |
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Components |
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-Supramolecule #1: Class4_80S_with_only_60S
Supramolecule | Name: Class4_80S_with_only_60S / type: complex / ID: 1 / Parent: 0 |
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Source (natural) | Organism: Rattus (rat) |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Buffer | pH: 7.4 |
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Vitrification | Cryogen name: ETHANE |
-Electron microscopy
Microscope | FEI TITAN KRIOS |
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Image recording | Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 50.0 e/Å2 |
Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.75 µm / Nominal defocus min: 1.25 µm |
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |